The long-term goal of this proposal is to develop PT523 [Nalpha-(4-amino-4-deoxypteroly)-Ndelta-hemiphthaloyl-L-ornithine], a water soluble, non-polyglutamtable analog of methotrexate (MTX), for the treatment of advanced lung cancer. Lung cancer is among the most commonly occurring malignancies in the world and is one of the few that continues to show an increasing incidence. In the US, lung cancer is the leading cause of cancer death. Most patients with lung cancer present with locally advanced or metastatic disease at the initial diagnosis, and the current standard care for these patients is systemic chemotherapy with a two-drug combination regimen that included a platinum agent. Although the current combination chemotherapy reduces the rate of death attributed to lung cancer, disease progress is inevitable and the dose-limiting toxicities restrict their use. Therefore, development of new combination therapies that aims to inhibit tumor growth and progression while sparing normal cells will improve the quality of life of lung cancer patients. The Specific Aims are: (1) To determine the effect of PT523 in combination with various cytotoxic and targeted therapeutic agents on tumor cell proliferation in vitro. Lung cancer cell lines will be treated with PT523 and various anti-cancer agents alone and in combination for 48 to 96 hours. The cytotoxic agents will include currently approved chemotherapy drugs for lung cancer and targeted therapeutic agents will include small molecule inhibitors for EGFR. The EGFR expression profile in various lung cancer cell lines will be determined by quantitative PCR and immunocytochemistry. The effect of various treatments on lung cancer cell proliferation will be quantified by MTT assay. The agents that work synergistically with PT523 in inhibition of cell growth will be determined. (2) To determine the efficacy of PT523 in combination with various cytotoxic and targeted therapeutic agents on tumor growth in xenograft models. Before evaluating the antitumor activity of PT523 with different agents, the maximum tolerated dose (MTD) of various agents alone or in combination will be determined in athymic mice. The growth inhibition activity of PT523 in combination with various agents will be evaluated in athymic mice bearing human lung carcinomas as s.c. transplants. The agent(s) that are most efficacious in inhibition of tumor growth and best tolerated in combination with PT523 will be selected.